CN106821987A - A kind of liposome and preparation method and application for carrying phenolic hydroxy group insoluble drug - Google Patents

Abstract

The invention provides a novel liposome and its preparation method and application. With phospholipids and polyethylene glycol 15-hydroxystearate as main materials, most insoluble drugs containing phenolic hydroxyl groups can be entrapped therein, increasing Drug solubility. And it can carry two insoluble drugs containing phenolic hydroxyl groups at the same time, which is suitable for combined drug use. At the same time, the liposome has various particle sizes, meets the passive targeting requirements of various organs, and has a sustained release effect. In addition, compared with traditional liposomes, the new liposome has the advantages of simple and cheap preparation process, more types of drug loading, better stability, etc., and has broad application prospects.

Description

Liposome loaded with insoluble drug containing phenolic hydroxyl group, preparation method and application thereof

technical field

The invention belongs to the field of pharmaceuticals, and relates to a liposome and a preparation method and application thereof. Specifically, the liposome can carry insoluble drugs containing phenolic hydroxyl groups in most structures.

Background technique

As we all know, the poor water solubility of drugs has always hindered the effective delivery of drugs. Research data show that up to 40% of candidate compounds that have been screened through high-throughput and have potential application prospects are difficult to dissolve in water, which hinders their development and application. Insoluble drugs have many disadvantages in the application of disease treatment, such as: poor water solubility will hinder the absorption of drugs, resulting in reduced bioavailability of drugs, especially oral drugs; insoluble drugs may cause vascular obstruction when administered intravenously. Deposition occurs in local tissues, causing various diseases. However, hydrophobicity is an inherent characteristic of many active substances, because its lipophilicity facilitates the drug to penetrate the cell membrane, allowing more drugs to reach the interior of the cell and exert a therapeutic effect on a specific target. Therefore, improving the solubility of poorly soluble drugs while retaining their therapeutic activity is a difficult and hot topic in the field of pharmacy today.

At present, the methods to improve the solubility of poorly soluble drugs mainly include:

One is to add acid or alkali to adjust the pH of the drug solution so that the drug can form soluble salts. Patent document CN201010537215.9 provides a method for adding organic amines to increase drug solubility, which is simple and easy to implement and is suitable for industrial production. However, after salt formation, the therapeutic activity of the drug may be affected. Moreover, after the salt-forming drug enters the human body, due to internal environmental factors such as temperature, pH, inorganic salts, and plasma proteins, the solubility of the drug may be reduced again, posing a serious safety hazard.

The second is to make insoluble drugs into prodrugs. Although this method can significantly improve the stability of the drug and make it difficult to change after entering the human body, it will destroy the original structure of the drug at the same time and may change its drug activity.

The third is to prepare insoluble drugs into new preparations such as clathrates, solid dispersions, nanoparticles, liposomes or emulsions. The advantage of this method is that while increasing the solubility of the drug, the structure of the drug is completely preserved without affecting the activity of the drug. Moreover, some dosage forms have a slow-release effect, which improves the curative effect and reduces toxic and side effects. Patent document CN201310165276.0 provides a solid dispersion of insoluble drugs and its preparation method. In this invention, insoluble genistein and other polyphenol hydroxyl drugs are prepared into solid dispersions by melting method and polymer materials, which significantly improves the drug dissolution rate and solubility. Patent document CN201010023013.2 provides a method for preparing microcapsules of insoluble drugs. The microcapsules have the advantages of increasing drug solubility and controlling drug release rate. Patent document CN201510870096.1 provides a nano-skeleton system and a preparation method for encapsulating insoluble drugs. This nano-skeleton system has the advantages of improving drug solubility, dissolution rate and bioavailability.

Liposome is a new type of pharmaceutical preparation, the main component is phospholipid, which can be used as a carrier for insoluble drugs. Phospholipids are endogenous components of the human body with good biocompatibility and safety. After intravenous administration, liposomes are swallowed by the reticuloendothelial system in the body, so that the drug is mainly distributed in organs such as the lung, liver, spleen and bone marrow. The particle size is different, and the main distribution organs are also different. In addition, if there is a tumor in the body, the liposome will also stay in the tumor site through the EPR effect, thereby achieving passive targeting. Patent document CN200610021277.8 provides a preparation method and application of a liposome, which can entrap insoluble drugs and magnolol for tumor targeting therapy. However, at present, most liposomes are based on phospholipids and cholesterol. The types and quantities of insoluble drugs that this liposome can carry are very limited, and the stability of drug-loaded liposomes is not good. , Often there will be drug precipitation within a few days, which greatly limits its application.

The present invention aims to design a new type of liposome, which does not use the traditional combination of phospholipids and cholesterol. This liposome can entrap most or a large class of insoluble drugs, increase drug solubility, and has excellent Excellent stability, no drug precipitation and no obvious particle size change within a week or longer. After a series of screening, the inventors found that liposomes prepared from phospholipids and polyethylene glycol 15-hydroxystearate (trade name: Kolliphor HS15) can meet the above requirements. In addition, the inventors further discovered during the research process that this new type of liposome can simultaneously entrap two poorly soluble drugs in it, and has excellent stability. As we all know, combined drug use is a hot spot in the field of pharmaceutical research at present, and has its unique advantages: on the one hand, combined drug use can allow multiple drugs to act on the same patient site at the same time, improving drug targeting; The complementary characteristics of these compounds can play a synergistic or additive effect, and can achieve the purpose of synergizing and reducing toxicity, so as to better exert the drug effect. This discovery greatly expands the application of the novel liposome of the present invention, and researchers can freely choose to use it to entrap one drug or simultaneously entrap two drugs for combined drug therapy. In addition, the liposome also has a certain slow-release effect, prolongs the action time of the drug, and reduces toxic and side effects. Moreover, liposomes with different particle sizes can be obtained by changing the feeding ratio, so that they can be distributed to different organs. According to different diseased organs, liposomes with suitable particle size are selected to carry corresponding therapeutic drugs to achieve the purpose of targeted and precise treatment.

Contents of the invention

One of the objectives of the present invention is to provide a novel liposome, which does not use the combination of phospholipid+cholesterol commonly used in ordinary liposomes as a material.

One of the objectives of the present invention is to provide a liposome that can carry most or a large class of insoluble drugs, increase the solubility of insoluble drugs, and expand the application of liposomes.

One of the objectives of the present invention is to provide a liposome that can simultaneously entrap two poorly soluble drugs, which can be used as a carrier for combined medication.

One of the objectives of the present invention is to provide a liposome with sustained release effect.

One of the objectives of the present invention is to provide a liposome with passive targeting effect. Liposomes with different particle sizes can be obtained by changing the prescription, which can be passively targeted to different tissues and organs.

The inventors found by studying the structure of insoluble drugs that the insoluble drugs containing phenolic hydroxyl structure can generate an interaction force with the positively charged quaternary ammonium nitrogen in phospholipids due to the negative charge properties of phenolic hydroxyl groups, so that the drugs and phospholipids form complexes . In addition, the phenolic hydroxyl structure can also form hydrogen bonds with the hydrophilic head of phospholipids, further increasing the possibility of using phospholipids to support insoluble drugs containing phenolic hydroxyl structures. However, phospholipids alone cannot stably entrap poorly soluble drugs. After carrying out the screening of various surfactants, the inventors found that the liposomes prepared by using polyethylene glycol 15-hydroxystearate in combination with phospholipids can encapsulate most insoluble drugs containing phenolic hydroxyl structures. Loaded in it, and the stability is excellent. Therefore, this new type of liposome with stronger drug-loading ability was creatively invented. This liposome has the advantages of various types of drug-loaded, good stability and wide application.

The inventors also found that the liposome can simultaneously contain two or more insoluble drugs containing phenolic hydroxyl structures, and has the advantages of strong drug loading capacity and can be used for combined administration.

The invention provides a kind of liposome taking phospholipid and 15-hydroxypolyethylene glycol stearate as material; Phospholipid and 15-hydroxypolyethylene glycol stearate mass ratio are preferably 20:1～1: The larger the proportion of 20,15-hydroxy polyethylene glycol stearate, the smaller the liposome particle size. Phospholipids or polyethylene glycol 15-hydroxystearate cannot be used alone to entrap drugs stably.

The particle size of the liposome is preferably 40nm-200nm.

The liposome has a drug loading of 0.1% to 20%, preferably 2% to 10%.

One of the objectives of the present invention is to provide the application of the above-mentioned liposome. This liposome can not only be used to contain insoluble drugs containing phenolic hydroxyl structures, but also can be used for combined drug use, sustained release of drugs, passive targeted therapy of tumors and other tissues, etc.

One of the objectives of the present invention is to provide a preparation method of the above-mentioned liposome.

As one of specific embodiments, the preparation method of liposome of the present invention is as follows:

(1) Mix and dissolve phospholipids, polyethylene glycol 15-hydroxystearate and drug in an organic solvent according to a certain mass ratio, and place them in a round-bottomed flask;

(2) Film formation by rotary evaporation, hydration by adding water, ultrasonic probe or high-pressure homogenization, and the product is obtained.

Preferably, the water includes deionized water, water for injection, physiological saline, 5% glucose solution and the like.

The phospholipids in step (1) are selected from natural soybean phospholipids, natural egg yolk phospholipids, hydrogenated phospholipids and synthetic phospholipids of different specifications from commercial sources, and can be selected from phospholipids S45, S75, S100, SPC, E80, EPCS, EPG, SPC of German Lipoid company -3, DSPE, DPPA, DSPA, DMPC, etc., phospholipid PC98-T,

PL-100M, HSPC, PGE, PGSH, etc., Korean Doosan Corporation phospholipid DS-PL95E, etc., preferably phospholipids E80, S100, PC98-T, EPCS, etc., are commonly used commercially available phospholipids.

The organic solvent in step (1) is preferably ethanol, methanol, dichloromethane, chloroform, acetone, etc. and their mixed solvents.

The mass ratio of phospholipids to polyethylene glycol 15-hydroxystearate in step (1) is preferably 20:1~1:20.

The drug loading in step (1) is preferably 2%~10%. Insoluble drugs containing phenolic hydroxyl groups suitable for the present invention include but are not limited to the following:

Antineoplastic drugs, including but not limited to, curcumin and its derivatives, resveratrol, honokiol, teniposide, temoporfin, daunorubicin, nororubicin, diethyl daunorubicin, Oxodiacyloxydaunorubicin, Zorubicin, Idarubicin, Arubicin, Amrubicin, Pirarubicin, Liurubicin, Medorubicin, Menoril, Nai Morubicin, rhodorubicin, detorubicin, ethorubicin, doxorubicin, epirubicin, aclarmycin, noramycin, stetemycin, mitoxantrone, pyrrole Anthraquinone, etoposide, lanreotide, vapreotide, edotretide, olivine, antramycin, hydroxycamptothecin, combretastatin A-4, etc.

Other drugs, including but not limited to, levodopa, dobutamine, silibinin, hydroxycoumarin, aspartocin, vapreotide, chloroquinaldol, ketamine, clioquinol , Diiodoquinoline, Tibuquin, Mehydroxyquine, Mebromoxyquine, Isoniahydrazone, Troglitazone, Hydroxybuzone, Acetaminophen, Salbutamol, etc.

Honokiol, curcumin, silibinin, resveratrol, pirarubicin or teniposide are preferred.

The combination of two drugs can be contained at the same time, including but not limited to, curcumin and its derivatives + honokiol, resveratrol + honokiol, teniposide + honokiol, daunorubicum Susin + honokiol, doxorubicin + honokiol, hydroxycamptothecin + honokiol, silibinin + honokiol, curcumin and its derivatives + resveratrol, tini Poside + silibinin, resveratrol + doxorubicin, silybin + resveratrol, etc.

The following combinations are preferred: honokiol+teniposide, curcumin+resveratrol, adriamycin+resveratrol, honokiol+resveratrol, honokiol+curcumin, etc.

Beneficial effect

(1) The liposome of the present invention has a large drug loading capacity, good stability, good safety and biocompatibility.

(2) The liposomes of the present invention have various particle sizes, and liposomes with a particle size of 40nm-200nm can be obtained by controlling the feeding ratio to meet different targeting requirements.

(3) The liposome of the present invention can entrap a large class of insoluble drugs containing phenolic hydroxyl structures, and has many types of drugs and a wide range of applications.

(4) The liposome of the present invention can simultaneously entrap two insoluble drugs containing phenolic hydroxyl structures, has a strong drug-loading capacity, and is suitable for joint administration.

(5) The liposome of the present invention has passive targeting. Liposomes of different particle sizes can be selectively passively targeted to organs such as bone marrow, liver, spleen, tumor and lung.

(6) The liposome of the present invention has a certain sustained-release effect.

(7) The preparation process of the liposome of the present invention is simple and easy to control, and is suitable for industrial production.

Description of drawings

Below, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:

Fig. 1 represents the transmission electron micrograph of liposome in embodiment 1 (magnification is 80,000 times).

Figure 2 shows the results of the in vitro release experiment of liposomes in Example 1.

Figure 3 shows the experimental results of tumor cell uptake of DiD-labeled liposomes.

Fig. 4 shows the in vivo distribution results of DiD-labeled liposomes.

Fig. 5 shows the safety evaluation results of liposomes of the present invention.

Fig. 6 shows the result of the drug effect of the liposome of the present invention used in combination.

Figure 7 shows the stability comparison results of liposomes of the present invention and commonly used liposomes.

Fig. 8 shows the comparative results of in vivo pharmacokinetics between liposomes of the present invention and commonly used liposomes.

detailed description

The following examples are to further illustrate the present invention, but in no way limit the scope of the present invention. The present invention is further described in detail below with reference to examples, but those skilled in the art should understand that the present invention is not limited to these examples and the preparation method used. Moreover, those skilled in the art can perform equivalent replacement, combination, improvement or modification of the present invention according to the description of the present invention, but these will all be included in the scope of the present invention.

Example 1

Dissolve 40mg of phospholipid E80, 25mg of polyethylene glycol 15-hydroxystearate and 6mg of honokiol in ethanol in a round-bottomed flask, rotatively evaporate to form a film, add normal saline to hydrate, and probe sonicate to obtain a thick film. Parkol liposome, particle size 84nm, intravenous injection for tumor targeting therapy.

Example 2

Dissolve 100mg of phospholipid S100, 5mg of polyethylene glycol 15-hydroxystearate and 3mg of curcumin in dichloromethane, place in a round bottom flask, rotary evaporate to form a film, add normal saline to hydrate, and homogenize under high pressure to obtain turmeric Vegetarian liposome, particle size 200nm, intravenous injection sustained release for inflammation treatment.

Example 3

Dissolve 5mg of phospholipid SPC, 100mg of polyethylene glycol 15-hydroxystearate and 2mg of silibinin in acetone, place in a round-bottomed flask, rotate to evaporate to form a film, add 5% glucose solution for hydration, and ultrasonically obtain the product Silibinin liposome, particle size 40nm, intravenous injection for the treatment of liver fibrosis.

Example 4

Dissolve 50mg of phospholipid E80, 15mg of polyethylene glycol 15-hydroxystearate and 3mg of resveratrol in ethanol in a round-bottomed flask, rotary evaporate to form a film, add normal saline to hydrate, and homogenize under high pressure to obtain white Veratrol liposome, particle size 85nm, intravenous injection for tumor targeting therapy.

Example 5

Dissolve 60mg of phospholipid S45, 60mg of polyethylene glycol 15-hydroxystearate and 3mg of pirarubicin in chloroform, place in a round-bottomed flask, rotatively evaporate to form a film, add normal saline to hydrate, and probe ultrasonically to obtain pirarubicin Bixing liposome, particle size 68nm, intravenous injection for tumor targeting therapy.

Example 6

Dissolve 40mg of phospholipid S75, 25mg of polyethylene glycol 15-hydroxystearate and 2mg of teniposide in a mixed solvent of methanol and acetone in a round-bottomed flask, rotary evaporate to form a film, add normal saline to hydrate, probe The teniposide liposome was obtained by ultrasound, with a particle size of 78nm, which was used for tumor targeting therapy by intravenous injection.

Example 7

Dissolve 20mg of phospholipid E80, 40mg of polyethylene glycol 15-hydroxystearate and 3mg of resveratrol in dichloromethane and place in a round-bottomed flask, rotatively evaporate to form a film, add 5% glucose solution for hydration, and hydrate under high pressure. Resveratrol liposomes were obtained from the quality, with a particle size of 65nm, which was used for tumor targeting therapy by intravenous injection.

Example 8

Dissolve 80mg of phospholipid S100, 10mg of polyethylene glycol 15-hydroxystearate and 6mg of curcumin in methanol and place in a round-bottomed flask, rotary evaporate to form a film, add 5% glucose solution for hydration, and probe sonication to obtain curcumin Liposome, particle size 110nm, intravenous injection for tumor targeting or inflammation therapy.

Example 9

Dissolve 50mg of phospholipid EPCS, 10mg of polyethylene glycol 15-hydroxystearate and 7mg of honokiol in ethanol in a round-bottomed flask, rotary evaporate to form a film, add normal saline for hydration, and homogenize under high pressure to obtain the Magnolol liposome, particle size 86nm, intravenous injection for tumor targeting therapy.

Example 10

Dissolve 10mg of phospholipid PC98-T, 100mg of polyethylene glycol 15-hydroxystearate and 4mg of silibinin in ethanol in a round-bottomed flask, rotary evaporate to form a film, add normal saline for hydration, and obtain the product by ultrasound Silibinin liposome, particle size 50nm, intravenous injection for the treatment of liver fibrosis.

Example 11

Dissolve 15mg of phospholipid E80, 60mg of polyethylene glycol 15-hydroxystearate and 3mg of pirarubicin in chloroform, place in a round bottom flask, rotatively evaporate to form a film, add normal saline to hydrate, and probe ultrasonically to obtain pirarubicin Bixing liposome, particle size 60nm, intravenous injection for tumor targeting therapy.

Example 12

Dissolve 70mg of phospholipid EPG, 7mg of polyethylene glycol 15-hydroxystearate and 2mg of teniposide in a mixed solvent of methanol and acetone in a round-bottomed flask, rotatively evaporate to form a film, add 5% glucose solution to hydrate , high-pressure homogenization to obtain teniposide liposomes, particle size 78nm, intravenous injection for tumor targeting therapy.

Example 13

Dissolve 4mg of phospholipid E80, 48mg of polyethylene glycol 15-hydroxystearate and 5mg of curcumin in methanol and place in a round bottom flask, rotary evaporate to form a film, add normal saline for hydration, and probe sonication to obtain curcumin lipid Body, particle size 48nm, intravenous injection for tumor targeting or inflammation therapy.

Example 14

Dissolve 15mg of phospholipid PL-100M, 75mg of polyethylene glycol 15-hydroxystearate and 4mg of honokiol in acetone in a round-bottomed flask, rotary evaporate to form a film, add normal saline for hydration, and homogenize under high pressure. Honokiol liposomes were obtained, with a particle size of 57nm, which was used for tumor targeting therapy by intravenous injection.

Example 15

Dissolve 75mg of phospholipid E80, 5mg of polyethylene glycol 15-hydroxystearate and 4mg of resveratrol in ethanol in a round-bottomed flask, rotatively evaporate to form a film, add 5% glucose solution for hydration, and probe sonication Resveratrol liposome, particle size 150nm, intravenous injection for tumor targeting therapy.

Example 16

Dissolve 60mg of phospholipid S100, 10mg of polyethylene glycol 15-hydroxystearate and 7mg of silibinin in dichloromethane, place in a round-bottomed flask, rotatively evaporate to form a film, add normal saline to hydrate, and homogenize under high pressure. Silibinin liposomes were obtained, with a particle size of 90nm, which was used for the treatment of liver fibrosis by intravenous injection.

Example 17

Dissolve 6mg of phospholipid E80, 90mg of polyethylene glycol 15-hydroxystearate and 3mg of pirarubicin in chloroform, place in a round bottom flask, rotatively evaporate to form a film, add normal saline to hydrate, and probe ultrasonically to obtain pirarubicin Bixing liposome, particle size 45nm, intravenous injection for tumor targeting therapy.

Example 18

Dissolve 12mg of phospholipid EPCS, 96mg of polyethylene glycol 15-hydroxystearate and 4mg of teniposide in a mixed solvent of methanol and acetone in a round-bottomed flask, rotary evaporate to form a film, add normal saline to hydrate, and pressurize Homogenized to obtain teniposide liposomes, particle size 55nm, intravenous injection for tumor targeting therapy.

Example 19

Dissolve 72mg of phospholipid E80, 6mg of polyethylene glycol 15-hydroxystearate and 5mg of resveratrol in methanol, put it in a round bottom flask, rotatively evaporate to form a film, add 5% glucose solution for hydration, and probe sonication Resveratrol liposome, particle size 130nm, intravenous injection for tumor targeting therapy.

Example 20

Dissolve 90mg of phospholipid S100, 5mg of polyethylene glycol 15-hydroxystearate and 4mg of honokiol in acetone and place in a round-bottomed flask, rotary evaporate to form a film, add 5% glucose solution for hydration, and homogenize under high pressure. Honokiol liposomes were obtained, with a particle size of 180nm, which was used for tumor targeting therapy by intravenous injection.

Example 21

Put 40mg of phospholipid E80, 25mg of polyethylene glycol 15-hydroxystearate, 3mg of honokiol and 3mg of teniposide in a mixed solvent of methanol and acetone in a round-bottomed flask, rotary evaporate to form a film, add Physiological saline hydration, probe ultrasound to obtain honokiol-teniposide co-loaded liposomes, particle size 90nm, intravenous injection for tumor-targeted synergistic drug delivery.

Example 22

Dissolve 50mg of phospholipid S100, 15mg of polyethylene glycol 15-hydroxystearate, 4mg of curcumin, and 4mg of resveratrol in ethanol in a round-bottomed flask, rotary evaporate to form a film, add normal saline for hydration, and pressurize The curcumin-resveratrol co-loaded liposome with a particle size of 100nm can be obtained by intravenous injection for tumor targeted synergistic drug delivery.

Example 23

Dissolve 60mg of phospholipid EPCS, 20mg of polyethylene glycol 15-hydroxystearate, 4mg of doxorubicin and 3mg of resveratrol in ethanol in a round-bottomed flask, rotary evaporate to form a film, add 5% glucose solution to hydrate , Probe ultrasound to obtain adriamycin-resveratrol co-loaded liposomes, particle size 95nm, intravenous injection for tumor-targeted synergistic drug delivery.

Example 24

Dissolve 100mg of phospholipid E80, 30mg of polyethylene glycol 15-hydroxystearate, 5mg of honokiol and 5mg of resveratrol in ethanol in a round-bottomed flask, rotatively evaporate to form a film, add normal saline to hydrate, Honokiol-resveratrol co-loaded liposomes were obtained by high-pressure homogenization, with a particle size of 110nm, which was used for tumor-targeted synergistic drug delivery by intravenous injection.

Example 25

Dissolve 70mg of phospholipid PC98-T, 20mg of polyethylene glycol 15-hydroxystearate, 4mg of honokiol and 3mg of curcumin in acetone and place in a round-bottomed flask, rotary evaporate to form a film, add normal saline to hydrate, Honokiol-curcumin co-drug-loaded liposomes were obtained after high-pressure homogenization, with a particle size of 105nm, which was used for tumor-targeted synergistic drug delivery therapy by intravenous injection.

The relationship between the feed ratio of experimental example 1 phospholipid E80 and 15-hydroxystearic acid polyethylene glycol ester and liposome particle size

By controlling the feeding ratio of phospholipid E80 and polyethylene glycol 15-hydroxystearate (represented by HS15 in the table), liposomes with different particle sizes can be obtained to meet different drug delivery requirements, as shown in Table 1. The results showed that the larger the proportion of polyethylene glycol 15-hydroxystearate, the smaller the liposome particle size.

Table 1. The relationship between feed ratio and particle size

Experimental example 2 Importance of combined use of phospholipid E80 and polyethylene glycol 15-hydroxystearate

The inventors investigated the use of phospholipid E80 or polyethylene glycol 15-hydroxystearate (represented by HS15 in the table) alone to prepare drug-loaded liposomes, as shown in Table 2. The results showed that the particle size and PDI of liposomes prepared by using phospholipid E80 or polyethylene glycol 15-hydroxystearate alone were larger and very unstable. Therefore, phospholipid E80 and polyethylene glycol 15-hydroxystearate must be used in combination in a certain ratio to prepare liposomes meeting the inventor's requirements.

Table 2. Preparation of drug-loaded liposomes using phospholipid E80 or polyethylene glycol 15-hydroxystearate alone

Experimental example 3 drug-loaded liposome stability investigation

The present invention selects the honokiol liposome in embodiment 1 as a model, and studies the stability when this novel liposome entraps a kind of insoluble drug; selects the drug-loaded liposome in embodiment 21 as a model , to study the stability of this new type of liposome when simultaneously loading two insoluble drugs. The prepared liposomes were stored at 4°C, room temperature and 37°C respectively, and the particle size was measured by sampling at regular intervals, and the changes in particle size were recorded, as shown in Table 3 and Table 4.

Table 3. Stability study of liposomes individually loaded with a drug

Table 4. Stability investigation of liposomes loaded with two drugs at the same time

The results show that the liposomes of the present invention entrap one or both drugs have excellent stability, there is no significant change in particle size within 10 days, and no drug precipitation, and the temperature has no significant impact on the preservation of liposomes .

The morphology of experimental example 4 liposomes, particle size measurement

The concentration of honokiol liposomes (drug+excipient) in Example 1 was diluted to 1 mg/ml, and the morphology and particle size of the liposomes were observed under a transmission electron microscope.

Fig. 1 is the transmission electron micrograph of honokiol liposome. The results showed that the appearance of honokiol liposomes was round and the particle size was uniform. Under this embodiment, the particle size of the honokiol liposome is about 80-100nm.

In vitro release of experimental example 5 honokiol liposome

2ml honokiol former drug solution (0.5mg/ml) and 2ml honokiol liposome suspension (0.5mg/ml, calculated by honokiol concentration) in Example 1 were placed in dialysis respectively in the bag (molecular cut-off 1000Da). Place the dialysis bag in a brown jar containing 100ml of PBS (pH7.4), and shake it on a constant temperature shaker (100rpm) at 37°C. Take 5.0ml of the dialysate outside the dialysis bag at regular intervals to measure the ultraviolet absorption value, and add 5.0ml of fresh PBS to the jar at the same time. The concentration of honokiol was calculated by substituting the UV absorption value into the standard curve, and then the release degree of honokiol original drug and honokiol liposome at each time point was calculated (n=3).

Fig. 2 is the in vitro release experiment result of honokiol liposome in embodiment 1. The results show that the honokiol liposome has better sustained-release effect than the original drug of honokiol in vitro, and the liposome of the present invention can be used for drug sustained release.

Experimental Example 6 Tumor Cell Uptake Test

Label liposomes with lipophilic fluorescent dye DiD, and trace the liposomes. The preparation method is similar to Example 1, specifically: 40 mg phospholipid E80, 25 mg 15-hydroxystearic acid polyethylene glycol ester and 0.6 mg DiD was dissolved in ethanol and placed in a round-bottomed flask. Rotary evaporation was used to form a film. Physiological saline was added to hydrate, and the probe was sonicated to obtain DiD-labeled liposomes (DiD-Lip), with a particle size of 82nm.

Inoculate mouse melanoma cells (B16F10) in a 12-well plate, ¹ ×105 cells per well, with RPMI-1640 medium (containing 10% fetal bovine serum, 50U/ml penicillin, 50U/ml streptomycin ) in a 37°C, 5% CO ₂ incubator overnight to achieve 80% monolayer coverage of the cells. Aspirate the medium, and add 1ml DiD original drug solution and DiD-Lip suspension to each well (dilute the concentration to 0.5μg/ml with the medium, and calculate according to the DiD concentration). Each of the original drug group and the liposome group had three replicate holes, and another three holes were set up as controls. After continuing to culture for 2 h, the medium was discarded, washed twice with PBS, and the cells were digested with trypsin. Digestion was terminated after 1 min, the cells were transferred to a 2ml centrifuge tube for centrifugation, the supernatant was discarded, resuspended in 300 μl PBS, and the fluorescence intensity of DiD was measured by flow cytometry.

Figure 3 shows the results of tumor cell uptake of DiD-Lip (**: p <0.01). The results show that the uptake of liposome by tumor cells is significantly higher than that of DiD original drug, and the liposome of the present invention has the potential as a tumor-targeting drug carrier.

Experimental example 7 in vivo distribution research

⁶ -week-old C57 mice were inoculated with B16F10 cells in the axilla (2 × 106 cells per mouse, dispersed in 0.2 ml PBS). When the tumor grew to about 200mm ³ (14 days after inoculation), the mice were divided into two groups: DiD original drug group and DiD-Lip group (the preparation method was the same as that in Experimental Example 6), with 3 mice in each group. After intravenous injection of the original drug and liposome respectively (administration dose is 10 μg/kg, calculated according to the concentration of DiD), they were executed 2 hours later, and the heart, liver, spleen, lung, kidney, and tumor were taken out, washed, and in vivo imager Each organ was semi-quantified, and the DiD content in the organ was determined.

Figure 4 shows the distribution results of DiD-Lip in vivo. The result shows that the distribution of the liposome in the tumor tissue is obviously higher than that of the original drug group, which further proves that the liposome of the present invention can be used as a carrier for tumor targeting therapy.

Experimental Example 8 Safety Evaluation

Ten male SD rats were randomly divided into 2 groups, 5 rats in each group. The two groups received intravenous injections of normal saline and liposomes (without drug loading, the prescription was the same as in Example 1, and the concentration was 50 mg/kg), once every 2 days for 4 consecutive weeks. During the administration period, the living conditions of the rats such as food intake, activity, and mental state were observed, and blood was collected 24 hours after the last administration to determine hematological indicators, including white blood cell count (WBC), red blood cell count (RBC) and platelet count (Plt). After sacrificing the rats, the heart, liver, spleen, lung, kidney and other organs were taken out, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned and stained with hematoxylin-eosin for histopathological examination under an optical microscope. The pathological changes of various organs of the two groups of rats were observed.

Figure 5 shows the safety evaluation results of liposomes (a: blood routine indicators; b: pathological sections of major organs, the scale represents 200 μm). The results showed that there was no significant difference between the hematological indexes of the liposome group and the normal saline group, indicating that the liposome had no effect on bone marrow hematopoiesis. In addition, the liposome has no obvious toxicity to major organs, which further shows that the liposome of the present invention has good safety.

Experimental example 9 liposome is used for combination medicine

The inventor selected two antitumor drugs, teniposide and honokiol, and entrapped them in liposomes separately and in the same liposome at the same time, and compared the antitumor effects of drug combination and drug alone, To explore the application prospect of this liposome in the field of combination medicine.

⁶ -week-old C57 mice were inoculated with B16F10 cells in the axilla (2 × 106 cells per mouse, dispersed in 0.2 ml PBS). When the tumor grew to about ^50mm3 (8 days after inoculation), the mice were divided into 5 groups: normal saline group (a), teniposide liposome group (b), honokiol liposome group (c ), teniposide liposome + honokiol liposome group (d) and teniposide + honokiol co-loaded liposome group (e), 3 rats in each group. Each group was given corresponding preparations, and then administered once every other day, and the administration was stopped after 6 administrations, and the experiment was stopped 21 days after inoculation. After the last administration, the mice were dissected, the tumors were removed, and the tumor size of each group was observed. The preparation process of each group of preparations is as follows:

Group a: normal saline;

Group b: Encapsulate teniposide into liposomes according to the prescription amount under Example 1, and the dosage is 10 mg/kg;

Group c: Honokiol is entrapped in liposome according to the prescription amount under Example 1, and the dosage is 10 mg/kg;

Group d: prepare teniposide liposomes and honokiol liposomes respectively according to the prescription amount under embodiment 1, and then co-administer after the two kinds of liposomes are mixed by volume ratio 1:1, administration The dosage is calculated according to the total drug dosage, which is 10mg/kg;

Group e: Teniposide and honokiol are co-encapsulated into the same liposome according to the prescription amount in Example 21 at a mass ratio of 1:1, and the dosage is calculated according to the total drug amount, which is 10 mg/kg .

The experimental results are shown in Figure 6. The results showed that the therapeutic effect of liposome-encapsulated two drugs in combination was significantly better than that of single-drug liposome. Moreover, it is more effective to entrap two drugs in the same liposome than to mix two single-drug liposomes. Therefore, the liposome of the present invention has great potential for combination medicine.

Comparative Experiment

In order to further demonstrate the superiority of the liposome of the present invention compared to the liposome commonly used at present, the inventor set up a comparative experiment. Patent document CN200610021277.8 provides a liposome, which is prepared from commonly used phospholipids and cholesterol, and added with PEGylated phospholipids, and also contains honokiol, which is in line with our comparison need. Therefore, the inventors used the liposome in the patent document CN200610021277.8 as a comparative preparation.

Comparison of the types of packaged drugs

The inventor selected several representative poorly soluble drugs, respectively entrapped them with the liposome of the present invention and the liposome in the patent document CN200610021277.8, and compared the entrapment capabilities of the two. The liposome of the present invention was prepared according to the prescription amount under Example 1 of the present invention; the comparative liposome was prepared according to the method under Example 1 in patent document CN200610021277.8, and the results are shown in Table 5.

It can be seen that the liposome of the present invention can entrap all selected drugs, the particle size and PDI are smaller, and the appearance is better; while the comparison liposome can only entrap honokiol better, Poor inclusion of other drugs of choice. The results show that the liposome of the invention can carry more types of poorly soluble drugs, and has a strong carrying capacity.

Table 5. Comparison of the types of entrapped drugs

2. Comparison of liposome stability

Prepare honokiol liposomes according to the prescription under Example 1 of the present invention and the prescription under Example 1 in patent document CN200610021277.8 respectively, store at room temperature, and take samples at regular intervals to measure the particle size. The ratio of the sampling particle size to the initial particle size was used to characterize the change in particle size, and the respective stability of the two liposomes was investigated, and the results are shown in Figure 7.

It can be seen that the particle size of the liposome of the present invention hardly changes within 10 days; while the particle size of the comparative liposome has been increasing, and the particle size has increased from about 100nm to about 300nm after 10 days. The results show that the liposome of the present invention has better stability.

In vivo pharmacokinetic comparison of liposomes

Honokiol liposomes were prepared according to the prescription under Example 1 of the present invention and the prescription under Example 1 in the patent document CN200610021277.8 respectively. Nine healthy male Wistar rats (180 ± 20g) were randomly divided into three groups of honokiol original drug group, comparison liposome group and liposome group of the present invention, with 3 rats in each group, and each preparation was given respectively, The dosage is calculated as 20mg/kg according to honokiol. At regular intervals after the administration, 300 μl of blood was taken from the orbit, placed in an EP tube containing 1% sodium heparin, and centrifuged at 6000 rpm for 5 minutes. Take 100 μl of supernatant, add 400ul of methanol to precipitate protein, vortex for 10min, then sonicate in water bath for 10min, centrifuge at 13000rpm for 10min, take the supernatant, and measure the plasma drug concentration at each time point by HPLC, the results are shown in Figure 8.

The results show that, without using PEGylated phospholipids, the long-circulation effect of the liposomes of the present invention is also slightly better than that of the comparison liposomes containing PEGylated phospholipids in the prescription, reflecting the superiority of the liposomes in long-term circulation performance, and the production cost is cheaper.

Claims (14)

1. A liposome containing a phenolic hydroxyl insoluble drug, characterized in that the preparation comprises: a phenolic hydroxyl insoluble drug, phospholipid, polyethylene glycol 15-hydroxystearate. 2. A liposome containing phenolic hydroxyl insoluble drugs, characterized in that the preparation comprises: two or more insoluble drugs containing phenolic hydroxyl groups, phospholipids, polyethylene glycol 15-hydroxystearate . 3. The liposome according to claim 1 or 2, characterized in that the mass ratio of phospholipids and polyethylene glycol 15-hydroxystearate is 20:1 ~ 1:20. 4. liposome according to claim 1 or 2, is characterized in that the drug loading of liposome is preferably 2%～10%. 5. liposome according to claim 1 or 2, is characterized in that, phospholipid is selected from the natural soybean phospholipid of commercial source different specification, natural egg yolk phospholipid, hydrogenated phospholipid and synthetic phospholipid, preferably, phospholipid is selected from phospholipid S45, S75, S100, SPC, E80, EPCS, EPG, SPC-3, PC98-T, PL-100M, HSPC, PGE, PGSH, DS-PL95E, DSPE, DPPA, DSPA, DMPC, etc., more preferably phospholipids E80, S100, PC98-T, EPCS. 6. The liposome according to claim 1 or 2, wherein the insoluble drug containing phenolic hydroxyl group is selected from antineoplastic drugs and other drugs containing phenolic hydroxyl group. 7. according to the described liposome of claim 1, it is characterized in that, the insoluble drug containing phenolic hydroxyl group is selected from the group consisting of antineoplastic drugs containing phenolic hydroxyl group, levodopa, dobutamine, silibinin, Hydroxycoumarin, aspartocin, vapreotide, chloroquinaldol, chloramphenicol, clioquinol, diiodoquinoline, tibuquine, medroxyquine, mebroxyroxyquine, isoniazone, Troglitazone, hydroxybuzone, acetaminophen, albuterol. 8. The liposome according to claim 2, wherein the two kinds of insoluble drugs containing phenolic hydroxyl groups are selected from the following combinations: antineoplastic drugs+antineoplastic drugs, antineoplastic drugs+non-antineoplastic drugs , and the combination of two non-antineoplastic drugs such as: levodopa + silibinin, dobutamine + silibinin, acetaminophen + salbutamol, ketamine + clioquinol, Diiodoquinoline + tibuquine, hydroxycoumarin + aspartocin, vapreotide + troglitazone. 9. according to the described liposome of claim 1 or 7, it is characterized in that, the preferred honokiol, curcumin, silibinin, resveratrol, pirarubicin or Teniposide. 10. according to the described liposome of claim 2 or 8, it is characterized in that, described two kinds of insoluble drugs containing phenolic hydroxyl group are preferably following combination: honokiol+teniposide, curcumin+resveratrol, Doxorubicin + Resveratrol, Honokiol + Resveratrol, Honokiol + Curcumin. 11. A method for preparing liposomes according to any one of claims 1-10, characterized in that (1) phospholipids, polyethylene glycol 15-hydroxystearate and insoluble drugs containing phenolic hydroxyl groups Mix and dissolve in an organic solvent according to a certain mass ratio and place in a round-bottomed flask; (2) Rotary evaporation to form a film, add solvent for hydration, probe ultrasonic or high-pressure homogenization. 12. preparation method according to claim 11 is characterized in that described organic solvent is selected from ethanol, methyl alcohol, methylene chloride, chloroform, acetone etc. and mixed solvent thereof, and the solvent of described hydration is selected from deionized water, Distilled water, normal saline, 5% glucose solution. 13. A liposome according to claim 1 or 2, characterized in that it can be used to increase the solubility of poorly soluble drugs, drug combination, drug sustained release and passive targeted therapy of tissues such as tumors, and has a long circulation role. 14. A use of the liposome according to any one of claim 2 or 8 or 10 in the preparation of a combined drug preparation.